Although the total amount of histone H3 associated with the viral genome remained fairly constant (Fig. ), we observed dynamic acetylation and methylation patterns of HCMV chromatin at different stages of the infectious cycle. The patterns fall into two groupings: immediate-early promoters versus all of the other promoters and nonpromoter regions (Fig. and ). At the start of infection, histone H3 at immediate-early promoters was hypomethylated (K9) and hyperacetylated (K9 and K14). In contrast, early and late promoters, as well as nonpromoter domains, contained hyper-methylated and hypo-acetylated H3 at 3 and 6 hpi. As the infection entered the late phase, the level of H3 methylation at the immediate-early promoter increased, whereas H3 methylation at the other promoters and nonpromoter regions was reduced. Late after infection all regions of the viral genome tested were associated with hyper-acetylated H3. The late hyper-acetylation of H3 was substantially blocked by cycloheximide or PAA treatment (Fig. ), indicating that the late modification requires continuing cellular and/or viral gene expression and viral DNA replication.
The key to the different patterns of methylation and acetylation at immediate-early compared to early and late promoters probably resides in their organization. Early and late promoters are relatively simple in terms or their constituent transcription factor binding sites, whereas immediate-early promoters are more complex. For instance, the MIEP promoter/enhancer region includes five CREB/ATF 19-bp repeat sites, four NF-κB 18-bp repeat sequences, three 21-bp repeats with Sp1, YY1 and ERF binding sites, three retinoic acid receptor response elements and two AP1 consensus sites (40
). Although individual binding sites or groups of sites, such as the full set of NF-κB (5
) or CREB/ATF (25
) sites, can be deleted with little apparent effect on function of the MIEP during viral replication in fibroblasts, mutation of Sp1 sites (22
) and deletion or substitution of substantial portions of the enhancer region reduce its activity (21
). Some of the cellular factors binding at the MIEP almost certainly direct histone modifying activities to the promoter/enhancer at the start of infection. For example, YY1 is known to bind the histone acetyltransferases and HDACs (62
), as well as histone methyltransferases (4
The level of acetylated H3 at immediate-early promoters is initially higher (3 and 6 hpi) before it is reduced at 12 hpi. We believe that this dynamic behavior is mediated by the viral IE2 protein. Relatively little IE2 is present at the start of the infection, but as it accumulates it binds to the CRS within the MIEP (1
) and recruits chromatin remodeling factors that silence transcription (50
). Consistent with this interpretation, the reduced acetylation of histone H3 at 12 hpi does not occur after infection with a mutant virus lacking a functional CRS, and MIEP H3 acetylation at 24 and 48 hpi is greater than in the wild-type virus (Fig. ). HDAC inhibitors also blocked the transient hypoacetylation at 12 hpi (Fig. ) with an increase in IE2 expression at this time (Fig. ). Inhibition of protein synthesis by treatment with cycloheximide also modulated acetylation at immediate-early promoters, inducing an increase in acetylation of H3 (Fig. ) with increased expression of immediate-early RNAs (data not shown). Since the drug was added before the start of infection, precluding the synthesis of IE2, part of the cycloheximide effect could result from the failure of IE2 to bind the MIEP and block its activity. However, the UL37 immediate-early promoter also undergoes transient hypoacetylation at 12 hpi (Fig. ), and was hyperacetylated in response to cycloheximide (Fig. ). We did not detect significant levels of IE2 at this promoter (Fig. ), arguing that additional mechanisms contribute to the changes in histone acetylation and elevated activity in the absence of protein synthesis. It seems likely that the cycloheximide effect on acetylation is due in part to differential stabilities of proteins involved in competing acetylation and deacetylation activities at the promoters. Possibly, a critical HDAC or factor that directs it to viral promoters has a shorter half-life than the activities promoting acetylation.
Why is the IE2-controlled hypoacetylation at the MIEP transient? It is likely that the dynamic effect results from the relative numbers of viral genomes versus IE2 molecules. Initially (3 and 6 hpi), IE2 has not accumulated to significant levels, most copies of the MIEP CRS are not occupied, histones at the MIEP are hyperacetylated and the promoter is highly active. At 12 hpi the ratio of IE2 to genomes is relatively high, because the MIEP has been maximally active, and its product, IE2, has accumulated, but the genome copy number has remained stable because DNA replication has not yet begun. As a consequence, IE2 occupies the CRS, recruits HDACs and inhibits MIEP activity. Then, less IE2 is produced as DNA replication begins and the genome copy number increases. At this point there is no longer sufficient IE2 available to occupy the available CRS motifs, and many copies of the promoter are again acetylated and highly active.
In contrast to its inhibitory behavior at the MIEP, IE2 can activate cellular and viral genes by recruiting histone acetyltransferases to cellular and viral promoters to initiate transcription (6
). Consistent with this earlier work, we detected IE2 at multiple early and late promoters after 24 hpi (Fig. ), a time when promoters were active (Fig. ) and contained hyperacetylated H3 (Fig. ). Our results reinforce the view that the ability of IE2 to bind to histone acetyltransferases contributes importantly to its transcriptional regulatory activities (38
). Importantly, IE2 is not the only factor that leads to increased H3 acetylation on HCMV chromatin, since nonpromoter regions of the genome where IE2 does not bind at a detectable level (Fig. ) are also acetylated after 24 hpi. It appears that replication of viral DNA is another important event that causes the acetylation of the viral genome, since treatment with PAA to block viral DNA replication caused hypoacetylation of H3 at viral promoters and nonpromoter regions (Fig. ). It is not clear why nonpromoter regions are acetylated. Perhaps the H3 acetylation late after infection is nonspecific and not functionally relevant. Alternatively, the modification might play a role in other events in the viral replication cycle such as DNA synthesis and packaging.
We tested the association of CREB/ATF with viral promoters because this cellular transcription factor has been shown to bind IE2 (28
). Even though IE2 was present at promoters other than UL37 (Fig. ), CREB/ATF was detected at only the MIEP and at the early UL112 promoter (Fig. ). As noted above, the activity of the MIEP within infected fibroblasts is unaffected when all five CREB/ATF-binding sites are deleted (25
), but it is likely that CREB/ATF will prove important for the MIEP function in different cell types or under different physiological conditions that have not yet been tested. CREB/ATF is important for efficient UL112 transcription (53
), where it might interact with DNA-bound IE2.
Our observations suggest that the virus exploits a competition between chromatin remodeling factors that acetylate histones to induce viral gene activation and factors that methylate histones to silence viral transcription. This competition is likely part of the mechanism by which the virus expresses its genes in an ordered cascade (Fig. ). Once the viral genome enters the nucleus and associates with histones, the H3 at immediate-early promoters is hyperacetylated and hypomethylated, arguing that they successfully recruit transcription factors, histone acetyltransferases, as well as other components of the basal transcriptional machinery components needed to initiate transcription. In contrast, early and late promoters are bound by hypoacetylated and hypermethylated H3, arguing that they either fail to recruit histone acetyltransferases or recruit HDACs, DNA, and histone methyltransferases. As viral immediate-early proteins accumulate, IE1 favors more global acetylation of the viral chromatin by blocking the activity of HDACs (46
) and IE2 recruits histone acetyltransferases (6
) to induce the activity of early and late promoters. As the infection progresses through the late phase (after 48 hpi), new virions are assembled. At this late time, the MIEP is bound to both acetylated (Fig. ) and methylated histone H3 (Fig. ). Perhaps these differentially modified histones reside at the same promoter. Alternatively, they could represent two distinct populations of HCMV chromosomes (Fig. ). The population with methylated H3 might be transcriptionally inactive because of IE2 binding to the CRS, whereas the population with acetylated H3 might be actively transcribed. The failure to deacetylate histones at the MIEP could result from limited steady-state levels of IE2 late after infection, and it could conceivably mark viral genomes for packaging.
FIG. 10. Model relating HCMV chromatin modifications to promoter activity. (A) At the start of infection, cellular factors (e.g., NF-κB and CREB) and viral factors delivered in virions (e.g., pUL82) bind to the complex immediate-early promoters (e.g., (more ...)
It is well established that DNA methylation, histone deacetylation, methylation of histone H3 at lysine 9 and its association with HP1 act as epigenetic markers to silence gene expression. Further, it is clear that factors controlling DNA and histone methylation physically and functionally interact (13
). What is not fully understood is the order in which the DNA and histone modifications take place, their interdependence, and how that interindependence varies among different loci. Experiments showing that a DNA methyltransferase complex recruits HDACs support a model in which DNA methylation leads to histone methylation (10
). On the other hand, methylated H3K9 has been shown to act as a marker that leads to DNA methylation (14
), and cells lacking H3K9 methyltransferases exhibit significantly diminished CpG methylation at some loci (23
). Regardless of the order in which DNA methylation, histone H3 methylation, and recruitment of HP1 occur, all of these combined processes are hallmarks of heterochromatin. Although the lack of methylation in bacterial DNA activates innate immunity (2
), it has been proposed that DNA methylation might serve as a cellular defense against infection by silencing the genome of an invading virus (11
). It is conceivable that the histone methylation we observed early after infection reflects an innate response to viral DNA.